Apparatus of irradiating biological specimens

ABSTRACT

A method of irradiating a biological specimen with ultraviolet, in particular a polynucleotide specimen selected from DNA or RNA, or optionally a protein. In the case where the specimen is DNA or RNA, or potentially proteins, the specimen is irradiated to cross-link the specimen to a substrate. In the case where the specimen is DNA, the specimen can also be irradiated to form thymine dimers. The method uses an apparatus which permits relatively precise control of the total ultraviolet dose received by the specimen, despite any changes of ultraviolet flux from the lamps which may occur from during any one experiment, or between a number of experiments. Thus, the method allows relatively highly reproducible results to be obtained.

This is a divisional of application Ser. No. 07/868,491, filed on Apr.17, 1991, now U.S. Pat. No. 5,288,647, which is a continuation ofapplication Ser. No. 07/189,285, filed May 2, 1988, now abandoned.

FIELD OF INVENTION

This invention relates to a method by which polynucleotide specimens(DNA or RNA), can be relatively automatically and safely irradiated,particularly for the purpose of fixing them to a substrate, and in thecase of DNA also forming thymine dimers.

BACKGROUND

A common technique for sequencing DNA is the Southern blot. Thistechnique involves transferring DNA fragments from agarose gels to amembrane, typically to a nitrocellulose membrane. The DNA fragment wouldthen be hybridized with labeled complimentary DNA probes. More recently,various nylon membranes have been used in place of nitrocellulose, sincenylon membranes generally bind DNA thereto better than nitrocellulose.Furthermore, it has also been found that DNA binding to a nylon ornitrocellulose membrane, could be considerably enhanced if the DNAfragments, following placement on the membrane, were irradiated withultraviolet light ("UV") at a peak emission of 254 nanometers ("nm").Such a technique has been described by Kandjian, Biotechnology, Vol. 5,p. 165-167 (Feb., 1987), as well as by Church et al., Proc. Natl. Acad.Sci. U.S.A., Vol. 81, p. 991-995 (Apr., 1984). The enhanced DNA bindingto the substrate permits any initial probe/DNA hybrids to be denatured,and the bound DNA fragments reprobed many times with probes of differentPG,3 sequences, without any significant loss of signal from any hybridsformed. Thus, the overall sensitivity of the Southern blot technique isenhanced.

It has also been recently known to irradiate DNA with 254 nm UV to formthymine dimers. Thymine dimer formation results in only partialdigestion of a DNA specimen upon exposure to a restriction enzyme, andthereby facilitates restriction site mapping of the specimen. Formationof thymine dimers by UV irradiation, with subsequent restriction sitemapping, is described by Whittaker et al., Gene, Vol. 41, p. 129-134(1986), and by Cleaver, Biochimica Et Biophysica Acta, Vol. 697, p.255-258 (1982). Furthermore, as suggested by Saito et al., TetrahedronLetters, Vol 22, No. 34, pp. 3265-3268 (1981), 254 nm UV can also beenused to irradiate thymine in the presence of primary amines, to produceN(1)-substituted thymines.

In practice, the above techniques are typically performed by animprovised arrangement, using one or more ultraviolet lightbulbspositioned in some convenient location in the laboratory, such as afumehood or lab benchtop. The DNA specimens (typically on a substrate)would simply be placed an appropriate distance from the UV lights suchthat the UV flux on the DNA is approximately that desired, and thelights manually energized and de-energized following elapse of anappropriate time.

There are several difficulties associated with the foregoing procedure.First, the extent to which any reaction occurs, for example,cross-linking of DNA to a substrate or formation of thymine dimers, isdependent upon the total energy received by the DNA specimen. This is afunction of the UV flux received by the specimen at any given time, overthe total time of exposure. However, the flux of a UV light source,typically a low pressure mercury lamp, is not constant over the life ofthe source. Furthermore, previously typically more than 1 UV lamp wasused simultaneously to irradiate the DNA specimen. Should a total orpartial failure occur in one of the lamps, the total flux received bythe specimen would drop by an unknown quantity. The foregoing factors,which lead to variations in flux received by a DNA specimen either overa given experiment, or from experiment to experiment, lead to a lack ofaccurately reproducible results. Another difficulty with the previouslyimprovised method, at least in the case where the DNA was to becross-linked to a substrate, was that often a laboratory technicianwould at best roughly guess the flux from the UV light source to beused, and then additionally roughly guess the time of exposure requiredto supply the required total energy (which may be available from areference, but was typically also estimated). This again led to resultswhich were not accurately reproducible. Furthermore, the typicalimprovised arrangement, could likely result in UV leakage. The dangersof shortwave UV are well known, and thus prior arrangements could resultin hazardous UV exposure to laboratory workers.

SUMMARY OF THE INVENTION

The present invention provides a method of irradiating a polynucleotidespecimen, typically for the purpose of cross-linking it to a substrate,or at least in the case of DNA, for alternatively forming thyminedimers. The term "polynucleotide" refers to either deoxyribonucleotide("DNA") or ribonucleotide ("RNA"). The method of the present inventionuses an apparatus suitable for irradiating polynucleotide specimens withan accurate amount of total UV dose, the accuracy of which is notaffected by a decrease in output from one or more ultraviolet lamps, soas to enhance the reproduceability of experimental results either duringa given experiment, or from experiment to experiment. The apparatusfurther provides a means by which a predetermined total energy can beprovided to a polynucleotide specimen for reliable cross-linking to asubstrate, or for thymine dimer formation. Alternatively a fixed totalUV dose can be selected, or if desired, simply a time of exposure can beselected. The apparatus further substantially eliminates any shortwaveUV exposure hazard to laboratory workers.

The method of the present invention then, involves irradiating apolynucleotide specimen, or optionally a protein specimen, withultraviolet ("UV"), using an apparatus comprising a housing with arepository for the specimen. An ultraviolet lamp fixture is fittedwithin the housing, which fixture receives at least one, and preferablya plurality, of ultraviolet lamps to illuminate the repository and henceany specimens placed thereon. An energy detector provides an indicationof the total ultraviolet dose received on the repository at any time. Acontrol is linked to the foregoing detector, and to the lamp receptacle.This control can de-energize the lamp fixture upon detection of aselected or predetermined total ultraviolet dose by the energy detector.Preferably, the control will de-energize the lamp fixture upon detectionof a predetermined ultraviolet dose of between approximately 1.0 to 2.0kiloJoules per square centimeter ("kJ/cm² "), and preferablyapproximately 1.6 kJ/cm². The dose of 1.6 kJ/cm² is preferably providedby a UV flux (i.e. intensisty) of about 4300 microwatts/cm² forapproximately 30 seconds.

A timer (the setting of which can be varied) is optionally provided,which is linked to the lamp receptacle. This timer can also de-energizethe lamp fixture upon a lapse of a selected period during which the lampfixture is energized.

The energy detector preferably has a sensor, typically in the form of aphotosensor, which provides a signal corresponding to the ultravioletflux on the repository. A detector circuit, which is also a part of theenergy detector, is connected to the foregoing sensor. This detectorcircuit provides an indication of the total ultraviolet dose received onthe repository based upon the signal which was received from the sensorover time.

The control circuit comprises at least one of a variable memory or afixed memory, and preferably both. The fixed memory retains a fixed,predetermined value corresponding to a predetermined quantity of energy.The variable memory on the other hand, allows for selection of avariable value corresponding to a selected quantity of energy. By afixed, first value is meant that this value either cannot be changedafter the apparatus has been assembled, or if it can, it cannot bechanged by the same means as the variable value and in fact is moredifficult to change. Typically, if the fixed value can be changed, theaccess to the means for changing it is limited in that such means isdisposed in an enclosed housing, or special tools are required tomanipulate such means. Furthermore, the fixed value is retained in thefixed memory even if the apparatus is turned off, or unplugged from anexternal power source. In the preferred case where the control circuithas both fixed and variable memories, a memory switch is additionallyprovided which can select either of the foregoing memories. Thus, bypositioning the memory switch as desired, the control circuit willdeenergize the lamp fixture upon detection of either the selected or thepredetermined UV dose.

In the preferred practice of the method, an apparatus is used in whichthe housing is substantially opaque to ultraviolet light, and inaddition to the repository, has a door which allows for access to aninterior of the housing. This embodiment is additionally provided with akeypad (which term as used in this application, includes suitableswitches, either pushbutton, dial, or any other type) on the outside ofthe housing and which is connected to the control circuit. The keypadcarries the memory switch previously mentioned, as well as keys by whichthe variable value in the variable memory can be selected. Additionally,a preferred feature, is a safety switch which de-energizes the lampfixture (assuming of course, that the lamp fixture is alreadyenergized), upon opening of the door. By such means, inadvertentexposure of an operator to the shortwave UV within the chamber, isavoided.

In the practice of the method of the invention, the specimen is placedinto the repository in the housing, and the operator then uses thekeypad to select whether the timer will deenergize the UV lampreceptacle, or whether the fixed memory or variable memory of thecontrol circuit will result in deenergizing of the UV lamp receptacle.In the event that a selected value of the variable memory, or the timer,will be used to deenergize the receptacle, then the corresponding valuesare entered into the keypad. The lamp fixture is then energized,preferably by a start switch also located on the keypad. Of course, ifthe apparatus includes the preferred safety switch, then the door mustfirst be closed before the lamp fixture can be energized.

An apparatus suitable for use in the practice of the method of thepresent invention, is also provided.

DRAWINGS

Embodiments of the invention will now be described with reference to theaccompanying drawings in which:

FIG. 1 is a perspective view of a UV irradiating apparatus suitable forpracticing the method of the present invention;

FIG. 2 is a view of the apparatus of FIG. 1, which view is similar tothat of FIG. 1 except with portions cut away to reveal interiorcomponents thereof;

FIG. 3 is a block diagram illustrating the circuitry used in theapparatus of FIGS. 1 and 2;

FIGS. 4A to 4D are overlapping portions of a part of the circuitry ofthe apparatus of FIGS. 1-3;

FIGS. 5A to 5D are overlapping portions of a second part of thecircuitry of the apparatus of FIGS. 1-3.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Referring to the drawings, the UV irradiating apparatus which is used inthe practice of the present invention, is generally numbered 2, and hasa substantially UV opaque chamber 8 which is essentially closed.However, chamber 2 is accessible through door 18 by gripping handle 20.A lower surface 9 of chamber 8 acts as a repository for a specimen,preferably a polynucleotide, which is typically disposed on a substrate.A lamp fixture 10, in the form of five pairs of lamp sockets 11 (onlyone pair of which is shown in FIG. 2), is disposed on an upper side ofchamber 8. Each pair of opposed sockets 11 is designed to receive wellknown elongated low pressure mercury UV lamps, such as lamp 12illustrated in FIG. 2, preferably one which has a maximum intensity ofemission of 254 nm. Chamber 8 is surrounded by a outer housing 4 to forma compartment 16 in which is carried the electronic circuits ofapparatus 2.

The electronic circuits for apparatus 2 are shown in generalized form inthe block diagram of FIG. 3, with portions and additional elements shownin more detail in the schematic of FIG. 4. Referring primarily to FIG.3, a power input line 30, which simply may be a cord to plug into astandard 110 or 220 volt line, is connected to a low voltage powersupply 32 the output of which is delivered through line 34 to thevarious circuits as required. Line 30 is also connected to lamp fixture10 through a relay 36 or equivalent. Relay 36 is de-activated (i.e.,turned to the "off" position to de-energize fixture 10) through line 37,or a door switch 22, or through timer 46 or a control circuit 42. Thus,when switch 22 is open (i.e., door 18 of apparatus 2 is open), relay 36is deactivated to deenergize fixture 10. Relay 36 is activated (i.e.turned to the "on" position to energize fixture 10) through line 47connected to a keypad 48 which is mounted outside housing 4.

In particular, line 47 is connected to start/reset button 54 on keypad48. Likewise, timer 46 and control circuit 42 are also connected tokeypad 48. A key 56 selects timer 46 as the means by which relay 36 isdeactivated, and hence by which fixture 10 is deenergized, followingelapse of a set time entered by keys 52 on keypad 48 after thestarts/reset button 54 is pushed. Keys 58, 60 on the other hand, selecta variable memory 44 or a fixed memory 45, respecively of controlcircuit 42 as the means which will deactivate relay 36. Selecting fixedmemory 44 results in relay 36 being deactivated, and hence fixture 10being deenergized, when the total energy which is received by thespecimen being irradiated (again, preferably a polynucleotide), asdetermined by photosensor 40 and a control circuit 48, corresponds tothe predetermined value stored in variable memory 44. Alternatively, ifkey 60 is used to select variable memory 45, then a value can be storedin memory 45 through keys 52 on keypad 48, which will correspond to aselected total energy as determined by detector circuit 38. A displaypanel 50, typically a light emitting diode display, indicates theselection of keys 56, 58, 60 and the entries made on keys 62.

It is to be noted that the value in fixed memory 45 is predetermined bya manufacturer or a service person. This is accomplished through dipswitches shown in FIGS. 5A and 5D, which are part of the fixed memory.Since the fixed memory value is more difficult to alter than thevariable memory value, requiring access into compartment 16, and sinceit is retained in fixed memory 45 even if apparatus 2 is disconnectedfrom a power line, it is a predetermined value.

A detailed schematic of most of the components of a circuit whichcorresponds to the block diagram of FIG. 3, is provided in FIG. 4A-4D,and 5A-5D. In those Figures, block A represents a light emitting diode("LED") control circuit. Block B is a current, to voltage, to frequencyconverter, which serves as energy detector 38, previously described. Abeeper represented by C, additionally provides an audible indicationwhen the UV dose equals that which has been selected or predetermined,or the elapsed time is equal to that selected, and the ultraviolet lampsare turned off. Timer 46 is essentially represented by block D. Block Eis a control for relay 36, which responds then to the timer 46, controlcircuit 42, and door switch 22. A keypad decoder F serves toappropriately decode input on keypad 48. A non-numeric decoder G decodesnon-numeric input on keypad 48, in particular the selection of thereset/start switch, the selection of the fixed or variable memory, andthe selection of the timer. Block H forms part of fixed memory 45, andallows the predetermined value to be varied (in binary decimal code)through use of two sets of dip switches shown. Input storage andscroller, I, holds all digit values, including those for the fixed andvariable memories, and the timer.

In practice of the method of the present invention using the apparatus 2described above, it will be assumed that line 30 has already beenconnected to a suitable source of power, and that suitable UV lamps arealready mounted in sockets 11. A specimen, typically DNA or RNA on asubstrate, is placed on floor 9 of chamber 8, and door 18 is thenclosed. The user then decides if the UV irradiation is to be basedsolely on time or total UV dose, and if the latter, if a selected valueis to be entered for the total UV dose or if the predetermined valuewhich corresponds to the value stored in fixed memory 44 is to be used.If time is to be selected, switch 54 is pressed, and the desired totaltime entered by keys 52 on keypad 48. If a selected value of total UVdose is desired, then key 58 is pushed, and the desired total UV dose isentered by keys 52. If the predetermined total UV dose is desired, thenkey 60 is simply pressed. Following the foregoing operations, key 54 ispressed to start UV irradiation of the specimen. If any incorrect keysare pressed in the foregoing operation (and this can be monitored on thedisplay 50), then key 54 can simply be pressed again and the operationrepeated.

When key 54 is pressed to start UV irradiation, it will be noticed thatnothing will happen unless door switch 22 is closed (i.e. door 18 isclosed). Assuming door switch 22 is closed, then UV lamp fixture 10 willbe energized for the selected total time, the selected total UV dose, orthe predetermined UV dose, depending upon the selection made by keys 56,58, and 60, as previously described. When either a selected orpredetermined total UV dose are selected by the user, energy detector 38provides a continuous indication to control circuit 42 of the total UVdose received on the floor 9 of chamber 8, based on the signal receivedfrom photodetector 40 adjacent floor 9. When the total UV doseindication of energy detector 38 corresponds to the predetermined orselected value (again, depending on the user's selection as described),relay 36 will be de-activated to de-energize UV lamp fixture 10 and turnoff the UV lamps. Of course, if a desired UV exposure time has beenselected by the user, then timer 46 will de-activate relay 36.

It will be seen from the above, that should the output of a UV lampdrop, or should a lamp totally fail, during a particular experiment, orover a number of experiments, the total energy received by a specimenwill still be relatively accurately determined by the apparatus. Thus,if either the selected or predetermined total UV dose values areselected, reproducible results are obtained regardless of variations inUV flux from the UV lamps. Furthermore, a user is protected from UVexposure by chamber 8 and door switch 22.

With regard to the predetermined value stored in the fixed memory, ithas been found that for the purposes of cross-linking a polynucleotidespecimen to a nylon or nitrocellulose substrate generally, that a totalUV dose of 1.6 kJ/cm² produces good results, even with variations in thesubstrate and other conditions. This corresponds to a UV flux in theapparatus 2 of about 4300 microwatts per square centimeter, for 30seconds.

Variations and modifications to the embodiments of the inventiondescribed above, are of course possible. Accordingly, the invention isnot limited to those specific embodiments described.

                  TABLE 1                                                         ______________________________________                                        KEYPAD ENCODER                                                                PCB wiring board                                                              Bill Of Materials                                                             Item  Quantity  Reference    Part                                             ______________________________________                                        1     7         U4,U5,U6,U7, MM74HC173N                                                       U10,U11,U12                                                   2     1         U8           CD4514BE                                         3     1         U9           CD4078BE                                         4     2         SW2,SW3      SW SPST DIP-8                                                                 CT2066                                           5     1         U2           MM74HC74N                                        6     2         C1,C2        0.10 MFD/50V                                                                  ECQ-V1H104JZ                                     7     2         U15,U14      MM74HC244N                                       8     1         U13          MM74HC04N                                        9     2         R1,R2        RPACK, 10.0K                                                                  9810E13A1002GL002                                10    1         U1           MM74C922N                                        11    1         U3           MM74HC125N                                       12    1         SW1          KEYPAD                                           ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        UV EXPOSURE UNIT, CONTROLLER                                                  Bill Of Hateriali                                                             Item  Quantity  Reference     Part                                            ______________________________________                                        1     1         C6            1.0 MFD/50V                                                                   ECQ-V1H105JZ                                    2     3         C8,C9,C12     10 MFD/25V                                                                    ECS-F1EE106K                                    3     1         C7            1000 pF/50V COG                                                               21RR510                                         4     4         U18,U19,U20,U22                                                                             MM74HC192N                                      5     1         U21           CD4060BE                                        6     3         U23,U24,U31   MM74HCOON                                       7     2         U25,U28       MM74HC100N                                      8     2         U26,U27       MM74HC04N                                       9     2         Q1,Q2         2N2907A                                         10    2         R12,R15       100 ohm                                                                       298J250                                         11    1         R16           1.5K ohm                                                                      29SJ250                                         12    1         C10           100 pF COG                                                                    21RD610                                         13    3         C11,C13,C14   0.1 MFD/50V                                                                   ECQ-V1H104JZ                                    14    1         U30           MM74HC123N                                      15    2         R14,RI3       39K ohm                                                                       29SJ250                                         16    1         SW4           SPST                                            17    2         R8,R5         20K ohm pot                                                                   EVM-CEGAO1B24                                   18    1         R7            470K ohm                                                                      29SJ250                                         19    4         R6,R3,R18,R19 10K ohm                                                                       29SJ250                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        20     2         R4,R11       1K ohm                                                                        29SJ250                                         21     1         R10          10 meg ohm                                                                    29SJ250                                         22     1         R16          330 ohm                                                                       29SJ250                                         23     1         BZ1          BEEPER                                          24     1         C4           *8 pF COG                                       25     1         C5           *24 pF COG                                      26     1         R9           330K ohm                                                                      29SJ250                                         27     4         D3,D1,D2,D4  1N4148                                          28     1         R17          5.1K ohm                                                                      29SJ250                                         29     1         U16          OP-90GP                                         30     1         X1           32768Hz, XTAL                                                                 X32768-ND                                       31     1         U17          AD637JH                                         32     1         DS1          DISPLAY, LED                                    33     1         U29          ICM7217AIPI                                     ______________________________________                                    

We claim:
 1. Apparatus for exposing a polynucleotide specimen to UVradiation, comprisinga UV-opaque chamber defining a region into which asubstrate comprising a polynucleotide specimen can be placed; a UV lampfixture disposed inside said chamber; a UV sensor disposed at a fixedposition inside said chamber; a first circuit coupled to said sensorcapable of indicating a total UV dose received at said region; and asecond circuit coupled to said first circuit and to said lamp fixture,capable of de-energizing said lamp fixture; wherein said second circuitcomprises (1) a first memory storing a first value corresponding to apredetermined total UV dose, and (2) a second memory storing a secondvalue corresponding to a total UV dose which may be selected by anoperator of the apparatus; wherein said first memory retains a valuecorresponding to a total UV dose between about 1.0 kJ/m² and about 2.0kJ/m².
 2. Apparatus as in claim 1, wherein said chamber comprisesa firstregion where said repository is disposed; and a second region in whichsaid UV lamp fixture is disposed.
 3. Apparatus as in claim 1, whereinsaid chamber comprisesa first region where said repository is disposed;and a second region in which said UV sensor is disposed.
 4. Apparatusfor exposing a polynucleotide specimen to UV radiation, comprisingaUV-opaque chamber; means for supporting a substrate comprising apolynucleotide specimen in said UV-opaque chamber; means for retrievingfrom a memory either a first value indicating a predetermined desired UVdose effective for crosslinking said specimen or a second valueindicating a predetermined desired UV dose which may be selected by anoperator, said second value being effective for crosslinking saidpolynucleotide specimen; means for irradiating said chamber with UVradiation; means for limiting a total UV dose to said first or secondvalues of said desired UV dose, comprising (1) a UV sensor disposed at afixed position in said chamber in a location where said sensor iscapable of receiving an accurate measure of UV applied to said specimen,(2) a first circuit coupled to said sensor capable of determining atotal UV dose received at said region, and (3) a second circuit coupledto said first circuit and to said means for irradiating, capable ofde-energizing said means for irradiating.
 5. Apparatus as in claim 4,wherein said UV radiation is between about 200 nanometers and about 290nanometers.
 6. Apparatus suitable for irradiating a polynucleotidespecimen, comprisinga chamber having a repository for a substratecomprising a polynucleotide specimen; an ultraviolet lamp fixturedisposed within the chamber, which fixture can receive at least oneultraviolet lamp to illuminate said repository; an energy detectordisposed in said chamber which can provide an indication of a totalultraviolet dose received on said repository, said energy detectorhaving (a) a sensor disposed inside said chamber, in a location wheresaid sensor is capable of providing a signal corresponding to anultraviolet flux on said specimen on the repository, and (b) a detectorcircuit connected to said sensor, so as to provide an indication of atotal ultraviolet dose received at said specimen on said repository, inresponse to the signal received from the sensor over time; a controlcircuit connected to said detector circuit and said lamp fixture,capable of de-energizing said lamp fixture upon detection of a totalultraviolet dose, said control circuit having (a) a fixed memory whichretains a first value corresponding to a fixed, predetermined totalultraviolet dose, (b) a variable memory which allows for the selectionof a variable, second value corresponding to a variable, selected totalultraviolet dose, and (c) a memory switch which engages either saidfixed or said variable memories, such that said control circuit willde-energize the lamp fixture upon detection of either said predeterminedor said selected total ultraviolet dose, respectively, said first andsecond values being effective for crosslinking said polynucleotidespecimen.
 7. Apparatus as in claim 6, additionally comprising a timerlinked to said lamp fixture, which timer is capable of deenergizing saidlamp fixture upon elapse of a preselected period during which the lampfixture is energized.
 8. Apparatus as in claim 6, wherein said UVradiation is between about 200 nanometers and about 290 nanometers. 9.Apparatus suitable for irradiating a polynucleotide specimen,comprisinga chamber having a repository for a substrate comprising apolynucleotide specimen; an ultraviolet lamp fixture disposed within thechamber, which fixture can receive at least one ultraviolet lamp toilluminate said repository; an energy detector disposed in said chamberwhich can provide an indication of a total ultraviolet dose received onsaid repository, said energy detector having (a) a sensor disposed insaid chamber, capable of providing a signal corresponding to anultraviolet flux on the repository, and (b) a detector circuit connectedto said sensor, so as to provide an indication of a total ultravioletdose received at said repository, in response to the signal receivedfrom the sensor over time; a control circuit connected to said detectorcircuit and said lamp fixture, capable of de-energizing said lampfixture upon detection of a total ultraviolet dose, said control circuithaving (a) a fixed memory which retains a first value corresponding to afixed, predetermined total ultraviolet dose, (b) a variable memory whichallows for the selection of a variable, second value corresponding to avariable, selected total ultraviolet dose, and (c) a memory switch whichengages either said fixed or said variable memories, such that saidcontrol circuit will de-energize the lamp fixture upon detection ofeither said predetermined or said selected total ultraviolet dose,respectively; wherein said fixed memory retains a value corresponding toa total ultraviolet dose between approximately 1.0 to 2.0 kJ/m².